Cellular glutathione peroxidase (GPX1), a major selenium (Se)-containing protein in the body, has been widely considered an antioxidant enzyme. Most striking, we have found that although the enzyme protects against oxidative stress mediated by reactive oxygen species (ROS) in vivo and in vitro, knockout of GPX1 actually renders mouse hepatocytes highly resistant to apoptosis and protein nitration induced by 0.4 mM peroxynitnte (OONO-, PN), a potent reactive nitrogen species (RNS). Because this promoting role of GPX1 in the PN-induced oxidative stress is so fascinating, we propose to determine the underlying biochemical mechanism, signal pathway, and metabolic relevance. Our long-term goal is to elucidate the physiological function of GPX1 gene expression in Se nutrition and human health. We will conduct eight experiments to achieve three specific aims. First, we will use both ROS generators and scavengers as well as glutathione synthesis modulators to determine whether moderate elevation of intracellular ROS and glutathione duplicates the GPX1 knockout effect on the PN-induced oxidative stress in mouse hepatocytes. Second, we will use specific inhibitors to test whether the GPX1 action in these cells is signaled by the cytochrome c/caspase 3, poly(ADP-nbose) polymerase (PARP), and mitogen-activated protein kinase (MAPK) pathways. Last, we will find out if and how GPX1 promotes oxidative stress induced by endogenous PN generated from acetaminophen metabolism in mouse hepatocytes and liver. Key assays will be oxidative injuries including DNA fragmentation or strand breaks and protein nitrotyrosine formation, apoptotic signaling including cytochrome c release and activation of caspase 3, PARP, and MAPK, and cell death or histopathology. Our results will unveil a novel function of GPX1 and Se, and help define their dual role in coping with ROS and RNS. These findings may not only enhance our understanding of the mechanisms of Se in preventing cancer, viral infection and chronic diseases, but also lead to fundamental changes in the current theory and application of antioxidants, and shed new light on pathogeneses of many ROS/RNS-related diseases. Therefore, this research will make significant contribution to guiding the optimal use of Se and GPX1 mimic to improve the US public health.